Disrupting cells and/or tissue in solid state in presence of solid denaturing substance

ABSTRACT

A method for disrupting frozen cells and/or tissue in a solid, frozen state in the presence of a solid denaturing substance is provided. The method comprises providing frozen cells and/or tissue in frozen solid form, adding thereto the solid denaturing substance in sold form, and applying mechanical force thereto for disruption of the frozen cells and/or tissue. The frozen cells and/or tissue include muscle cells and/or muscle tissue, wheat cells and/or wheat tissue, and maize cells and/or maize tissue. Further, the solid, denaturing substance in solid form is a crystalline substance and can be selected from urea, thiourea, guanidium chloride, guanidium thiocyanate and ammonium sulfate. Also the solid, denaturing substance in solid form is added to the frozen cells and/or tissue in frozen form in an approximately 1 to 20 fold (w/w) excess.

BACKGROUND OF THE INVENTION

The invention relates to a method for disrupting biological material, inwhich the biological material is disrupted in the solid state and in thepresence of a solid, denaturing substance.

When proteins, nucleic acids, fatty acids or other cell constituents areto be recovered, the cells have to be disrupted. A variety of methodsand apparatus for disrupting cells have been developed, since the cellsof various organisms differ in their behavior and, in some cases, canonly be disrupted with difficulty. Also, the individual organisms orcells differ greatly with regard to the quality of the disruption. Aparticular problem when disrupting cells is the simultaneous liberationof degrading enzymes such as nucleases, proteases, lipases orglucosidases. In general, specific inhibitors are added to the batch tosuppress such activities.

Usually, the cells are disrupted by means of ultrasound, French-press,high-pressure homogenizer or X-press while being suspended. To recoverproteins, protease inhibitors such as, for example, PMSF, EDTA orleupeptin are generally added. However, the fact that the proteaseactivity cannot always be suppressed sufficiently constitutes adisadvantage of these methods.

Solid cell material may also be comminuted in a mortar while coolingwith liquid nitrogen, or in a vibration grinding mill (see, for example,Hess B. & Brand, K. (1983) Cell and Tissue Disintegration. GeneralAspects. In Methods Enzym. Anal., Third Ed., Eds. Bermeyer, H. U. VCI,Weinheim, FRG, Vol. 2, 26-30). However, the fact that the protein yieldsis relatively low compared with the other methods constitutes adisadvantage of this method.

To recover nucleic acids, the cells are generally disrupted byhydrolyzing the cell wall by means of lysozyme in the presence of SDS.The proteins are generally hydrolyzed by proteinase K. However, the factthat nucleic acids can only be recovered with difficulty fromlysozyme-resistant cells constitutes a disadvantage of this method.

SUMMARY OF THE INVENTION

Object of the present invention was therefore to find a method which iswidely applicable and allows cell constituents to be recovered in highyields.

Surprisingly, it has now been found that biological material can bedisrupted readily in the solid state and in the presence of a solid,denaturing substance without degrading enzymes being activated.

Subject-matter of the present invention is therefore a method fordisrupting biological material, in which the biological material isdisrupted in the solid state and in the presence of solid, denaturingsubstance.

In a preferred embodiment, the solid, denaturing substance is acrystalline, preferably a crystalline organic, substance. Examples ofespecially preferred substances are urea, thiourea, guanidiniumhydrochloride, guanidinium thiocyanate or ammonium sulfate. Thedisruption may also be carried out in the presence of various denaturingsubstances.

In general, the denaturing substance is employed in an approx. 1- toapprox. 20-fold (w/w) excess, preferably in an approx. 1- to approx.10-fold (w/w) excess and in particular in an approx. 1-fold (w/w)excess. It is furthermore advantageous for the biological material to bedeep-frozen and preferably to remain deep-frozen during the disruptionmethod according to the invention. Suitable for this purpose in anadvantageous manner is, for example, liquid nitrogen.

In general, the biological material is disrupted by grinding, preferablyin the presence, of a grinding ball.

Suitable as biological material is any biological material such as, forexample, animal, human or plant cells, cell aggregates, tissues oranimal, human or plant material. Also suitable are microorganisms suchas fungi, bacteria, yeasts, protozoa or algae. Further suitable examplesare E. coli, streptomycetes, Acremonium, Tetrahymena, Euglena, maize,wheat, muscle tissue or Actinoplanes.

To subsequently isolate the cell constituents such as proteins, nucleicacids (DNA, RNA), fatty acids, carbohydrates or the like after thedisruption, the disrupted biological material is dissolved in a suitablebuffer. The buffers which are customary for the cell type in questionmay be used for this purpose. The final concentration of urea isgenerally approx. 1 to approx. 10 M, preferably approx. 1 to approx. 5 Mand especially approx. 4 M. It is advantageous to remove the cell debrisand other solid constituents, for example by centrifugation, before thedesired cell constituents are isolated by the methods known to theskilled worker.

DETAILED DESCRIPTION OF THE INVENTION

The method according to the invention is described hereinbelow ingeneral terms and with reference to examples:

Usually, the organism or microorganism in question is grown in standardmedia known to the skilled worker. Thereafter, the cells can beharvested, for example by means of centrifugation. Then, they areusually washed and can be frozen for storage purposes. Plants can begrown on standard soils in the light or in the dark. Plant material isobtained, for example, by cutting of leaves, stalks or roots, which areusually immediately shock-frozen, for example in liquid nitrogen.Finally, the material can be comminuted mechanically by grinding in anitrogen-cooled mortar.

The deep-frozen cell pellet, or the comminuted material, is thenintroduced into a shaking container consisting of, for example, teflonand cooled, for example using liquid nitrogen. The pellet is treatedwith, for example, urea in the abovementioned ratio. Advantageously, agrinding ball is then added, and the batch is shaken, for example in alaboratory vibration grinding mill, for example Dismembrator U, BraunMelsungen, Melsungen, FRG. Usually, the pellet is ground slowly in thepresence of the urea by grinding at, for example, 2600 rpm, to give apowder. Advantageously, the batch is cooled with liquid nitrogen, andthe procedure is repeated until a fine powder is obtained. After thedisruption, the powder obtained is dissolved in a suitable buffer to theabovementioned final concentration of urea in order to isolate the cellconstituents. The cell debris and other solid components areadvantageously removed by centrifugation. The supernatant contains thedesired cell constituents.

And important advantage of the method according to the invention is thatcell constituents such as, for example, proteins, but also seaweed oil,can be recovered in high yields and in a simple manner, in particularfrom cells which are difficult to disrupt. In particular, it waspossible to recover high-molecular weight (>20 kb) DNA was recovered inan advantageous fashion, for example from Actinoplanes, which arenormally difficult to disrupt when using, for example, lysozyme. The DNArecovered in accordance with the invention is therefore alsoparticularly suitable for isolating large DNA fragments. Also,optimization of the growing conditions for the microorganisms isgenerally not necessary in the method according to the invention.

The examples which follow are intended to illustrate the invention ingreater detail without limiting it thereto.

EXAMPLES

1. Disruption of E. coli

E. coli was grown at 37° C. in a shaking flask in 100 ml of LB medium(10 g/l Bacto tryptone, 5 g/l yeast extract (Difco, Detroid, Mich., USA)and 5 g/l NaCl) to an OD 600 nm of 15. The cells were harvested bycentrifugation for 15 minutes at 4000 rpm at 4° C. The supernatant wasdiscarded. The pellet was washed twice in 1 M KCl solution. Then, thepellet obtained was frozen in liquid nitrogen. 2 g of the frozen pellettogether with 2 g of urea were then transferred into a cooled teflonvessel in which a tungsten grinding ball (diameter 0.5 cm) was located,and ground for 1 minute in a vibration grinding mill (Dismembrator U,Braun Melsungen, Melsungen, FRG) at 2600 rpm. The vessel was cooled withliquid nitrogen, and the procedure was repeated several time(approximately 10 times). The protein yield amounted to 4 mg/g powder(urea content in the powder: 50%).

To isolate the protein, 2 g of the powder were dissolved in 2 ml of 10mM Tris/HCl, 5 mM EDTA, 10 mM MgCl₂, pH 7.5. Then, the DNA was digestedfor 30 to 60 minutes by adding 10 units of benzonase or 10 μl ofDNase/RNase solution (10 mg/ml DNAseI, 4 mg/ml RNaseA in 10 mMTris/HCl₂, pH 7.5) at room temperature. The batch was then centrifugedat 14,000 rpm. The proteins were located in the supernatant and could beused in further experiments.

2. Disruption of Streptomyces griseus

Streptomyces griseus was grown for 2 days at 29° C. and 240 rpm in 50 mlof TSB (30 g/l tryptic soy broth) in a 1—1 baffle flask (Schott,Regensburg, FRG). The mycelium was harvested by centrifugation for 15minutes at 5000 rpm and 4° C. Then, the pellet was washed in 1 M KCl. 2g of the pellet together with 2 g of urea were transferred into a cooledteflon vessel in which a tungsten grinding ball (diameter 0.5 cm) waslocated, and ground for 1 minute in a vibration grinding mill(Dismembrator U, Braun Melsungen, Melsungen, FRG) at 2600 rpm. Thevessel was cooled with liquid nitrogen, and the procedure was repeatedseveral times (approximately 10 times). The protein yield amounts to 5mg/g powder (urea content in the powder: 50%).

3. Disruption of Acremonium chrysogenum

Acremonium chrysogenum was grown for 3 days in minimal medium (30 g/lglycose, 3 g/l NaNO₃, 1 g/l K₂HPO₄, 0.5 g/l MgSO₄, 0.5 g/l KCl, 0.01 g/lFeSO₄) at 25° C. and 220 rpm. The cells were harvested by centrifugationfor 15 minutes at 5000 rpm and 4° C. Then, the pellet was washed in 1 MKCl. 2 g of the pellet together with 2 g of urea were transferred into acooled Teflon vessel in which a tungsten grinding ball (diameter 0.5 cm)was located, and ground for 1 minute in a vibration grinding mill(Dismembrator U, Braun Melsungen, Melsungen, FRG) at 2600 rpm. Thevessel was cooled with liquid nitrogen, and the procedure was repeatedseveral times (approximately 10 times). The protein yield amounted to4.5 mg/g powder (urea content in the powder: 50%).

4. Disruption of Tetrahymena thermophiles

Tetrahymena thermophiles was grown for 2 days in PPYS medium (10 g/lprotease peptone No. 3, 1 g/l yeast extract (Difco, Detroid, Mich., USA)and 1 ml/l trace elements (10 g/l sodium citrate, 24.3 g/l ironchloride) at 25° C. and 80 rpm. The cells were harvested bycentrifugation at 2000 rpm and 4° C. The pellet was subsequentlydeep-frozen in liquid nitrogen. 2 g of the pellet together with 2 g ofurea were transferred into a cooled Teflon vessel in which a tungstengrinding ball (diameter 0.5 cm) was located, and ground for 1 minute ina vibration grinding mill (Dismembrator U, Braun Melsungen, Melsungen,FRG) at 2600 rpm. The vessel was cooled with liquid nitrogen, and theprocedure was repeated several times (approximately 10 times).

5. Disruption of Euglena gracilis, Tetraselmis specialis, T. Chui and T.coneolutae

Euglena gracilis was grown in medium 9 (1 g/l sodium acetate, 1 g/l meatextract, 2 g/l bacto tryptone, 2 g/l yeast extract, 0.02 g/l potassiumnitrate, 0.002 g/l ammonium phosphate, 0.001 g/l magnesium sulfate, 0.04g/l calcium chloride) and 30 ml of soil extract (300 g/l garden soilwhich had been boiled for 10 minutes, allowed to settle, then filteredand brought to pH 7.5 using a 10% strength sodium carbonate solution).

Tetraselmis specialis, T. chui or T. coneolutae were grown for 7 days at25° C. and 100 rpm in medium 460 with half the salt concentration (0.3g/l glucose, 0.01 g/l yeast extract (Difco, Detroid, Mich., USA), 11.74g/l sodium chloride, 5.315 g/l magnesium chloride, 1.96 g/l disodiumsulfate, 0.555 g/l calcium chloride, 0.33 g/l potassium chloride, 0.095g/l sodium hydrogen carbonate, 0.05 g/l potassium bromide, 0.015 g/lboric acid, 0.02 g/l SrCl₂, 0.005 g/l iron (III) chloride, 0.025 g/lammonium sulfate, 0.005 g/l dipotassium phosphate, 0.15 sodium glycerolphosphate, 3 g/l Tris buffer, 1.5 g/l glutamic acid) and 3 ml of a metalsolution (1 g EDTA, 0.05 g/l FeCl₃, 0.15 g/l MnCl₂, 0.01 g/l ZnCl₂,0.005 CoCl₂, 1 g/l H₃BO₃, brought to pH 6.5 with NaOH) and 1 ml ofvitamin solution (0.003 g/l biotin, 1 g/l thiamine) in 100 ml in a500-ml flask. The cells were harvested by centrifugation for 30 minutesat 2000 rpm and 4° C. Then, 0.5 g of moist biomass was treated with 0.5g of urea and the mixture was transferred into a cooled Teflon vessel inwhich a tungsten grinding ball (diameter 0.5 cm) was located, and groundfor 1 minute in a vibration grinding mill (Dismembrator U, BraunMelsungen, Melsungen, FRG) at 2600 rpm. The vessel was cooled withliquid nitrogen, and the procedure was repeated several times (approx.10 times).

To recover the oil, the powder was treated with 4 ml of LM mixture(toluene/ethanol 1:1) and the mixture was incubated for 90 minutes at40° C. with stirring. Then, the mixture was centrifuged for 30 minutesat 5000 rpm, and the upper, organic phase was removed. Then, the residuewas treated with another 4 ml of LM mixture, shaken and centrifuged for30 minutes at 5000 rpm. Again, the upper phase was removed and combinedwith the first organic phase. Then, the collected organic phases wereconcentrated in a rotary evaporator. The flask now contained the pureseaweed oil.

6. Disruption of Maize

Maize seeds (Zea mays cv. Felix) were sown in moist vermiculite soil andgrown for 6 days at 30° C. in the light or in the dark (etiolated). Thesoil was always kept moist. The aerial parts of the plants were snippedoff using scissors and immediately cooled in liquid nitrogen. Then, theplant material was divided into smaller sections with the aid of asurgical blade or else ground in a nitrogen-cooled mortar. However, theplant material may also be employed uncomminuted. Then, 2 g of plantmaterial were treated with 2 g of urea and transferred into a cooledTeflon vessel in which a tungsten grinding ball (diameter 0.5 cm) waslocated, and ground for 1 minute in a vibration grinding mill(Dismembrator U, Braun Melsungen, Melsungen, FRG) at 2600 rpm. Thevessel was cooled with liquid nitrogen, and the procedure was repeatedseveral times (approximately 10 times). The protein yield amounted to7.5 mg/g powder (urea content in the powder: 50%).

7. Disruption of Wheat

Wheat (Triticum aestivum cv. Ralle) seeds were sown in moist vermiculitesoil and grown for 6 days at 30° C. in the light or in the dark(etiolated). The soil was always kept moist. The aerial parts of theplants were snipped off using scissors and immediately cooled in liquidnitrogen. Then, the plant material was divided into smaller sectionswith the aid of a surgical blade or else ground in a nitrogen-cooledmortar. However, the plant material may also be employed uncomminuted.Then, 2 g of plant material were treated with 2 g of urea andtransferred into a cooled Teflon vessel in which a tungsten grindingball (diameter 0.5 cm) was located, and ground for 1 minute in avibration grinding mill (Dismembrator U, Braun Melsungen, Melsungen,FRG) at 2600 rpm. The vessel was cooled with liquid nitrogen, and theprocedure was repeated several times (approximately 10 times). Theprotein yield amounted to 7.5 mg/g powder (urea content in the powder:50%).

8. Disruption of Muscle Tissue

Muscles from rats or mice were frozen in liquid nitrogen immediatelyafter the animals had died. Then, 2 g of deep-frozen muscle were mixedwith 18 g of urea and transferred into a cooled Teflon vessel (volume 20ml) in which a tungsten grinding ball (diameter 1 cm) is located, andground for 1 minute in a vibration grinding mill (Dismembrator U, BraunMelsungen, Melsungen, FRG) at 2600 rpm. The vessel was cooled withliquid nitrogen, and the procedure was repeated several times (approx.10 times). After the disruption, the powder contained 10% protein.

9. Isolation of Chromosomal DNA From Actinoplanes utahensis

The cultures were grown in 300-ml Erlenmeyer flasks containing 50 ml ofR2YE medium [(Hopwood, D. A. et al. (1985) Genetic manipulation ofStreptomyces: a laboratory manual. John Innes Foundation, Norwich, UK]at 28° C. and 180 rpm (5 days). The cell pellet (2 g) was harvested bycentrifugation at 4° C., placed into a 5-ml disruption vessel (teflon)together with a tungsten carbite ball (diameter 0.5 cm) and 2 g of ureaand frozen in liquid nitrogen. Then, the cell pellet was homogenized for1 minute in the Dismembrator mill at 2600 rpm. After each cycle, thedisruption vessel was cooled in liquid N₂. The process was repeatedthree times. The cold homogenizate was then resuspended in 20 ml of TSEbuffer (TSE: 25 mM Tris/HCl; 25 mM EDTA, 10.3% sucrose pH 8.0) togetherwith 10 μg/ml RNAse. The solution was subsequently treated with 10 ml of2% SDS. Then, 7 ml of neutral CHCl₃/phenol (1:1) were added and thesample was mixed. The two phases were separated by centrifugation (4000rpm, 10 minutes), and approximately 15 ml of the aqueous upper phasewere removed carefully using a pipette. This was placed into a freshtube and extracted once by treating with 3 ml of CHCl₃. Afterrecentrifugation (4000 rpm, 10 minutes) and phase separation, the upperphase was treated with {fraction (1/10)} volume 3 M sodium acetate (pH5.2) and 0.8 volume of isopropanol and the DNA was precipitated. The DNAwas then harvested from this solution by centrifugation at 15,000 g (30minutes) or by fishing with a glass rod. The DNA was washed with 70%ethanol, then dried and taken up in TE buffer.

We claim:
 1. A method for disrupting frozen cells and/or tissue,comprising the steps of: (a) providing frozen cells and/or tissue infrozen solid form; (b) adding a solid denaturing substance in solid formto the frozen cells and/or tissue in frozen solid form; and (c) applyingmechanical force to disrupt the frozen cells and/or tissue in frozenform in the presence of the solid denaturing substance to release cellconstituents comprising protein, nucleic acids, fatty acids and/orcarbohydrates from the frozen cells and/or tissue without activatingdegrading enzymes.
 2. The method as claimed in claim 1, wherein thesolid, denaturing substance in solid form is a crystalline substance. 3.The method as claimed in claim 2, wherein the crystalline substance is acrystalline organic substance.
 4. The method as claimed in claim 1,wherein the solid, denaturing substance in solid form is selected fromthe group consisting of urea, thiourea, guanidium chloride, guanidiumthiocyanate and ammonium sulfate.
 5. The method as claimed in claim 1,wherein the solid denaturing substance in solid form is added to thefrozen cells and/or tissue in frozen solid form in an approximately 1-to approximately 20-fold (w/w) excess.
 6. The method as claimed in claim1, wherein the frozen cells and/or tissue in frozen solid form aredeep-frozen.
 7. The method as claimed in claim 1, wherein the frozencells and/or tissue in frozen solid form are disrupted at lowtemperature.
 8. The method as claimed in claim 7, wherein the frozencells and/or tissue in frozen solid form are disrupted while coolingwith liquid nitrogen.
 9. The method as claimed in claim 1, wherein thefrozen cells and/or tissue in frozen solid form are disrupted bygrinding.
 10. The method as claimed in claim 9, wherein the grinding iscarried out with a grinding ball.
 11. The method as claimed in claim 1,wherein the frozen cells and/or tissue in frozen solid form are selectedfrom the group consisting of muscle cells and/or muscle issue, wheatcells and/or wheat tissue, and maize cells and/or maize tissue.
 12. Themethod as claimed in claim 1, wherein the frozen cells and/or tissue infrozen solid form are selected from among microorganisms.
 13. The methodas claimed in claim 2, wherein the microorganisms are selected from thegroup consisting of fungi, bacteria, yeasts, protozoa and algae.
 14. Themethod as claimed in claim 1, wherein after the disruption of the frozencells and/or tissue in frozen solid form disrupted cells and/or tissueare subsequently suspended in a buffer.
 15. The method as claimed inclaim 14, wherein the buffer is added in an amount to provide a finalconcentration of the denaturing substance in the buffer of approximately1 to 10 M, 1 to 5 M or 4M.
 16. The method as claimed in claim 14,wherein after suspension of the disrupted cells and/or tissue in abuffer, the disrupted cells and/or tissue are centrifuged.